The Complement System
Constituting about 10% of the globulins in normal serum, the complement system is composed of many different proteins that are important in the immune system's response to foreign antigens. The complement system becomes activated when its primary components are fragmented and the fragments, alone or with other proteins, activate additional complement proteins resulting in a proteolytic cascade. Activation of the complement system leads to increased vascular permeability, chemotaxis of phagocytic cells, activation of inflammatory cells, opsonization of foreign particles, direct killing of cells and tissue damage. Activation of the complement system may be triggered by antigen-antibody complexes (the classical pathway) or for example, by lipopolysaccharides present in cell walls of pathogenic bacteria (the alternative pathway).
The Membrane Bound Complement Receptor Type 1
Complement receptor type 1 (CR1) is present on the membranes of erythrocytes, monocytes/macrophages, granulocytes, B cells, some T cells, splenic follicular dendritic cells, and glomerular podocytes. CR1 binds to the complement components C3b and C4b and has also been referred to as the C3b/C4b receptor. The structural organization and primary sequence of CR1 is known (Klickstein et al., 1987, J. Exp. Med. 165:1095-1112, Klickstein et al., 1988, J. Exp. Med. 168:1699-1717; Hourcade et al., 1988, J. Exp. Med. 168:1255-1270). It is composed of 30 short consensus repeats (SCRs) that contain 60-70 amino acids. In each SCR, 29 of the average 65 amino acids are conserved. Each SCR has been proposed to form a three dimensional triple loop structure through disulfide linkages with the third and first and the fourth and second half-cystines in disulfide bonds. CR1 is further arranged as 4 long homologous repeats (LHRs) of 7 SCRs each. Following a leader sequence, which is post-translationally removed, the CR1 molecule consists of the most N-terminal LHR-A comprising a C4b binding domain, the next two repeats, LHR-B and LHR-C comprising C3b binding domains, and the most C terminal LHR-D followed by 2 additional SCRs, a 25 residue putative transmembrane region and a 43 residue cytoplasmic tail.
CR1 is a member of a protein superfamily characterized by this SCR homology. Some superfamily members that have C3/C4 binding function include CR2, C4 binding protein, factor H, factor B, and C2, while proteins lacking this function include interleukin-2 receptor, .beta.2-glycoprotein I, C1r, haptoglobin .alpha. chain, and factor XIIIb.
CR1 is known to be a glycoprotein and its deduced amino acid sequence has 25 potential sites for N-linked glycosylation (amino acid consensus sequence NXS or NXT) in the extracellular region. Only 6-8 of the available sites were reported to be linked to oligosaccharides (Sim, 1985, Biochem. J. 232:883). A non-glycosylated form of CR1 has been produced in the presence of tunicamycin and showed reduced binding to iC3 (Lublin et al., 1986, J. Biol. Chem. 261:5736). The N-terminus of the glycoprotein appears to be blocked.
Thus far, four different CR1 allelic forms or allotypes have been identified, and differ in size by 30-50 kDa increments. The gene frequencies of these allotypes differ in the human population (Holer et al. 1987, Proc. Natl. Acad. Sci. USA 84:2459-2463). The F (or A) allotype is composed of 4 LHRs and has a molecular weight of about 250 kDa; the larger S (or B) allotype, with a molecular weight of about 290 kDa, contains a fifth LHR that is a chimera of the 5' half of LHR-B and the 3' half of LHR-A and is predicted to have a third C3b binding site (Wong et al., 1989, J. Exp. Med. 169:847). The smallest F' (or C) allotype, most likely arising from the deletion of LHR-B and one C3b binding site, has increased prevalence in patients with systemic lupus erythematosus (SLE) (Van Dyne et al., 1987, Clin. Exp. Immunol. 68:570; Dykman et al., 1983, Proc. Natl. Acad. Sci. USA 80:1698).
Soluble Complement Receptor Type 1
A naturally occurring soluble form of CR1 has been detected in the plasma of normal individuals and certain individuals with SLE (Yoon et al., 1985, J. Immunol. 134:3332-3338). Its characteristics are similar to those of erythrocyte (cell-surface) CR1, both structurally and functionally. Hourcade et al., 1988, J. Exp. Med. 168:1255-1270) also observed an alternative polyadenylation site in the human CR1 transcriptional unit that was predicted to produce a secreted form of CR1. The mRNA encoded by this truncated sequence comprises the first 8.5 SCRs of CR1, and encodes a protein of about 80 kDa which includes the C4b binding domain. When a cDNA corresponding to this truncated sequence was transfected into COS cells and expressed, it demonstrated the expected C4b binding activity but did not bind to C3b (Krych et al., 1989, FASEB J. 3:A368). Krych et al. also observed a mRNA similar to the predicted one in several human cell lines and postulated that such a truncated soluble form of CR1 with C4b binding activity may be synthesized in humans.
Several soluble fragments of CR1 have also been generated via recombinant DNA procedures by eliminating the transmembrane region from the DNAs being expressed (Fearon et al., Intl. Patent Publ. WO 89/09220, 10/5/89; Fearon et al., Intl. Patent Publ. WO 91/05047, 4/18/91). The soluble CR1 fragments were functionally active, bound C3b and/or C4b and demonstrated factor I cofactor activity depending upon the regions they contained. Such constructs inhibited in vitro the consequences of complement activation such as neutrophil oxidative burst, complement mediated hemolysis, and C3a and C5a production. A soluble construct, sCR1/pBSCR1c, also demonstrated in vivo activity in a reversed passive Arthus reaction (Fearon et al., 1989, 1991, supra; Yeh et al., 1991, J. Immunol. 146:250), suppressed post-ischemic myocardial inflammation and necrosis (Fearon et al., supra; Weisman et al., Science, 1990, 249:146-151) and extended survival rates following transplantation (Pruitt & Bollinger, 1991, J. Surg. Res 50:350; Pruitt et al., 1991 Transplantation 52; 868). Furthermore, co-formulation of the soluble product of vector sCR1 /pBSCR1c with p-anisoylated human plasminogen-streptokinase-activator complex (APSAC) resulted in similar anti-hemolytic activity as the sCR1/pBSCR1c product alone, indicating that the combination of the complement inhibitor sCR1 with a thrombolytic agent was feasible (Fearon et al., supra).
Each of International Patent Publication Number WO 89/09220, published Oct. 5, 1989,and WO 91/05047, published Apr. 18, 1991, are incorporated by reference herein in their entirety.